ABSTRACT: One-in-four adults hospitalized for community-acquired pneumonia (CAP) experience an adverse cardiac event. Clinical epidemiological studies, as well as those performed in mice, non-human primates, and with human autopsy samples indicate that Streptococcus pneumoniae (Spn), the leading cause of CAP, can invade the heart from the bloodstream and cause direct cardiotoxicity. Within the myocardium Spn cause focal areas of damage we have called microlesions and these disrupt contractility. One recent break-through in our understanding of Spn pathogenesis was the observation that pneumococci are taken up by cardiomyocytes and Spn kill these cells from within. What is more, the pore-forming toxin pneumolysin and Streptococcal pyruvate oxidase (SpxB) derived H2O2 were both requisite for cardiotoxicity. Herein, our goal is to gain an understanding of the events that take place within a cardiomyocyte immediately after Spn uptake. Along such lines, results from in vitro and in vivo experiments, including dual-species RNA sequencing of Spn- infected hearts, have revealed highly compelling connections between changes in carbon availability, H2O2 production, biofilm / cardiac microlesion formation, and pneumolysin production. Thus, we hypothesize that glucose restriction encountered by Spn within a cardiomyocyte, and again in cardiac microlesions, results in metabolic and gene expression changes that enhance bacterial cardiotoxicity. To test this hypothesis and learn how pneumolysin and H2O2 work together to kill cardiomyocytes we will: AIM 1: Determine how environmental glucose, metabolism, and virulence are interlinked. To elucidate the basis, extent, and consequences of these connections we will: 1) determine how purposeful shunting of pyruvate metabolism (by means of mutation) towards the production of acetate, lactate, and/or formate impacts gene expression under high and low glucose conditions; 2) identify how Spn gene expression changes in longitudinal fashion after bacterial uptake by a cardiomyocyte and how this is linked to changes in Spn metabolism; 3) determine the importance of metabolism-linked genes to Spn survival within a cardiomyocyte, killing of the cardiomyocyte, and the overall disease process. AIM 2: Determine how bacterial derived H2O2, together with pneumolysin, kills cardiomyocytes. SpxB derived H2O2 and pneumolysin are both required for Spn killing of cardiomyocytes; each alone is insufficient. To determine why we will: 1) determine how varying production of H2O2 and pneumolysin together modulate the form of cardiomyocyte death; 2) determine if H2O2 potentiates pneumolysin production, its release from Spn, or host cell membrane targeting; and, 3) determine if SpxB-derived H2O2 contributes to the ion dysregulation that has previously been implicated in pneumolysin-induced necroptosis. This aim, at its completion, will advance our understanding of how Spn kills host cells.